Method for dissociation of cells

ABSTRACT

Disclosed is a method for the dissociation of cells. Cells are processed under conditions of pH, temperature, and shear to thereby yield a mixture of cell wall ghosts and cytoplasm. Preferably, the cells are jet cooked at an alkaline pH to form an intermediate mixture, and the intermediate mixture is subsequently jet cooked. Generally, the cells become dissociated, whereby at least one separate cell wall component is substantially separate from the dissociated cell walls.

RELATED APPLICATION

This application claims the benefit of prior provisional applicationSer. No. 60/495,750, filed Aug. 15, 2003, the teachings of whichapplication are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention pertains to the dissociation of cells to obtain nutrientsand other commercially useful products therefrom.

BACKGROUND OF THE INVENTION

Yeast and yeast metabolites are widely used in an array of food and feedproducts. Baker's and brewer's yeast, for example, are excellent sourcesof nutrients and flavoring agents. Nutrients that are obtainable fromcells include insoluble and soluble cell wall polysaccharides,oligosaccharides, glucans, proteins, peptides, nucleotides, and thelike. Cells, in particular cell walls, are also thought to absorbpathogens and consequently to provide a measure of prophylaxis againstinfection.

Live cells, whole lysed cells, and cell fractions are of particularvalue in feed and pet food formulations. Lysed cells and cell fractionsare thought to contain many nutritive components in a form that isbio-available to the consuming animal. Live yeast cells are thought toaid in digestion in ways not fully understood at present. Whole deadcells, on the other hand, are not thought to be of particular nutritivebenefit, except possibly in ruminant animals. The digestive tract ofmonogastric animals is essentially unable to rupture the cell wall, andthus the majority of the dead cells pass through the digestive tract andare typically excreted whole, without releasing nutrients to the animal.

Consequently, if it is desired to obtain nutrients from dead yeastcells, generally it is necessary to rupture the walls of the cells toallow release of the nutrients. A number of methods are known forrupturing yeast cells, these including mechanical, hydrolytic andautolytic methods. Mechanical methods typically are employed insmall-scale laboratory applications. Conventional mechanical disruptionincludes presses, such as the French press; homogenizers; sonicdisruptors, and so forth. In a laboratory French press, for example,pressures as high 20,000 psi and high shear conditions are produced bypassing the cells through a small orifice. Other devices subject thecell to different stresses but provide the same result, that is, ruptureof the cell wall. For instance, another known apparatus, the beadbeater, contains ceramic or glass pellets that are used to crush, shearand fracture cells. Hydrolytic procedures employ enzymes, acid, oralkali to rupture the cell walls. Cell autolysis is a well-known processwherein the yeast cell is subjected to digestion by its own enzymes.

Heretofore, it is believed that it has been difficult to extractnutrients from cells on a commercial scale, particularly from dead yeastcells, in light of certain drawbacks with the foregoing conventionalmethods. Mechanical rupture is attractive because the cell constituentsare not contaminated with extraneous chemicals and additives. However,the costs associated with scaling-up and implementing such systems areconsiderable, as has been heretofore recognized. For instance, U.S. Pat.No. 5,756,135 issued to Seeley discusses some of the technological andeconomical challenges associated with commercial-scale production of awater insoluble yeast. Hydrolytic methods are more amenable to scale-up,but most such methods also have shortcomings such as high cost, longprocess time, or degradation/denaturation of specific nutrients.

Accordingly, most yeast cell hydrolyzates are produced commercially byautolysis. Yeast autolysis entails a slow reaction, however. Anautolysis reaction requires an operating temperature that ranges fromabout 40° C. to 60° C., typically temperatures of 50° C.-55° C. At theseor higher suitable temperatures, the reaction still requires asubstantial period of time ranging from several hours to days to obtaina suitable degree of digestion. In an effort to accelerate the autolysisreaction, the prior art has taught to employ plasmolyzing agents,examples of which include organic solvents, salts and hydrolytic enzymessuch as protease and lipases. Nonetheless, the autolysis reactionremains lengthy and commercially unwieldy.

A further drawback with autolysis is that the autolysis process isamenable only for use with living cells. Dead cells cannot be autolyzed.In recognition of this requirement, dedicated yeast manufacturers whodesire to autolyze the yeast cells are required to take steps topreserve cell viability. In other industries where substantialquantities of live yeast are produced as a by-product, such as thebrewing industry, live cells can be harvested economically and can besubjected to autolysis. However, certain industrial processes generate asubstantial quantity of dead yeast by-product that cannot be subjectedto autolysis. This is a particular problem in the production ofdistilled ethanol products, wherein the distillation process kills theyeast cells, thereby rendering the cells impossible to autolyze.

Accordingly, given the heretofore described drawbacks with mechanicaland hydrolytic methods, it is very difficult to produce acost-effective, high-volume yeast-derived feed or industrial productfrom such dead cells. In practice, the dead yeast cells themselves aresold as whole cells, typically into the ruminant animal feed markets.

It would be desirable to provide a method for disassociating yeast andother cells in a manner that allows for rupture of the walls of thecells to release the cell cytoplasm therefrom. It would be of particularbenefit for such method to be applicable to dead cells in addition tolive cells. Such method would find a particular applicability in thedistilled ethanol industry, but would also be useful in connection withnumerous other industries.

THE INVENTION

It has now been found that yeasts, fungi, bacteria, and other cells(including eukaryotic cells) may be processed to recover soluble orinsoluble cell components such as proteins, saccharides, peptides,lipids, glucans, and the like. Generally, the cells are processed by ashearing force in the presence of an alkaline pH and heat (i.e.temperatures above 25° C.).

In accordance with the invention, a method for dissociating cells isprovided. In one embodiment of the invention, conditions of pH, shear,and temperature suitable for dissociation of the cell are selected, theconditions being suitable for dissociation whereby at least one solubledissociated molecular cell wall component is substantially separablefrom the dissociated cells. The method is intended to at leastsubstantially completely dissociate the cell walls, but the cells arenot dissociated to such an extent that the molecular constituents of thecell walls are reduced to simple molecules. The soluble dissociated cellwall component may be separated from the dissociated cells.

In accordance with preferred embodiments of the invention, a method forproviding a mixture of cell wall ghosts and cytoplasm is provided. Themethod includes subjecting the cells to heat, shear, and pH underconditions sufficient to rupture the cell walls and to allow the releaseof cytoplasm therefrom while leaving a substantially intact cell wallghost. The method most preferably comprises jet cooking the cells. Inthe most highly preferred embodiments of the invention, the cells arejet cooked to form an intermediate product, and the intermediate productis subsequently jet cooked to form the mixture of cytoplasm and cells.The mixture thus formed may be spray dried or otherwise treated, such asby substantially separating the cell walls from the cytoplasm. An animalfeed may be prepared from the mixture or spray dried mixture thusformed.

BRIEF DESCRIPTION OF THE FIGURE

The FIGURE is a schematic illustration of a yeast cell wall dissociationmethod.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following paragraphs will focus primarily on the dissociation ofyeast cells, but it should be understood that the invention is notlimited thereto. Indeed, the invention is deemed to be applicable to anyprokaryotic or eukaryotic cells, in particular microbial cells, andespecially to yeasts. Other cells suitable for dissociation inconnection with the present inventive method include fungi, plant cells,spores, and like microorganisms. More generally, any cell that can be“harvested” to provide nutrients or other chemically useful materialscan be used in conjunction with the invention. If yeast is used, theyeast is preferably a strain of Saccharomyces cereviasiae, includingthose strains commercially sold as brewer's yeasts and baker's yeasts.The cells may be alive or dead, or mixtures of live and dead cells maybe employed. The yeast cells may be used as supplied from a commercialdistilling operation, or may be washed prior to use in conjunction withthe invention to remove bittering agents, fermentation insolubles, andthe like. It is contemplated that the yeast may include fibercarbohydrate, or other material from a commercial ethanol distillingoperation, and in some embodiments of the invention the yeast source maycomprise stillage. A preferred yeast source is spray dried yeast.

In accordance with the invention, the walls of cells are dissociated toyield cell wall components. The dissociation contemplates a wide rangeof dissociation of the cell walls, and the extent of dissociation may beselected by one of skill in the art. For instance, the cells as receivedmay contain impurities or non-native components that are bound viaelectrostatic forces (or even covalent bonds) to the cell walls. Thedissociation in some embodiments of the invention contemplates removalof these impurities or non-native components. In preferred embodimentsof the invention, the cell walls are partially disintegrated, such thatsome native cell wall components have been liberated from the molecularstructure of the cell walls, but that the cell wall ghosts are stilldiscernable as discrete entities under microscopic examination. It isthus contemplated that the ghosts may not be complete cell walls,inasmuch as some of the original components of the cell wall may havebecome dissociated from the remaining components of the cell wall. Anyportion of native cell wall components may be so liberated, such as 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, whereby in such embodiments,the cell wall ghosts are still discernable. In less preferredembodiments of the invention, the dissociation is completed to an extentsuch that the cell walls are substantially completely or fullydisintegrated, such that the cell walls are not visible as discreteentities under microscopic examination.

Generally, in accordance with the preferred embodiments of theinvention, the method for rupturing cells comprises subjecting the cellsto heat, pH, and shear under conditions sufficient to rupture the wallsof at least some of the plurality of the cells to allow cytoplasm to bereleased therefrom, thereby forming a mixture of ghosts and cytoplasm.The mechanism of action of the present invention is believed to benon-specific degradation or of the cells, whereby oligosaccharides (suchas mannanoligosaccharides) and glucans are released. Upon suchdegradation, the walls of the cells weaken eventually to the point ofcell wall rupture to thereby release the cytoplasm contained therein. Itis contemplated that conditions of temperature, pH, shear, and residencetime in a suitable apparatus will vary widely from species to species ofthe cell and will further vary depending upon the apparatus chosen.Generally, it is contemplated that the temperature employed will be in arange of from 140° to 160° C.

To hydrolyze the cell walls, a slurry of the yeast may be prepared byknown techniques, such as evaporation or known liquid-solid separationtechniques, or alternatively the yeast may be dried and subsequentlymixed with water to form a slurry. The solids content of the startingyeast slurry is preferably about 5 to 25% (w/v), preferably 10 to 20%,and more preferably 12 to 18%. It is desired to employ the solidscontent as high as is practicable, and an upper limit of 18 to 20% isdeemed most commercially practicable.

In carrying out the inventive method, the pH of the slurry of yeast isadjusted to any suitable pH, preferably a pH between 8.0 and 12.0, morepreferably 9.0 to 11.0, and most preferably 9.5 to 10.0, using an alkaliagent, most preferably a food-grade alkali such as sodium hydroxide,calcium hydroxide, or potassium hydroxide. The invention is not limitedto processing under alkaline conditions. In some embodiments, stronglyacidic conditions, preferably pH 0.5 to 3, and more preferably pH 1 to2, may be employed. The preferred acidifying agent is a food-grade acid,such as hydrochloric, phosphoric, sulfuric, or mixtures thereof. Becauseit is believed in most instances that an acid pH is far more aggressivethan the relatively mild alkaline conditions that may be employed foralkali hydrolysis of the yeast, alkaline conditions are preferred inconnection with the present invention.

The alkaline slurry of yeast is then subjected to shearing underconditions sufficient to rupture the walls of at least some of theplurality of the yeast cells to thereby release the cytoplasm.Generally, the cells may be subjected to a pressure of between 35 to 105psig at the conditions of temperature, pH, and shear heretoforediscussed. The cells are preferably subjected to such pressure for atime ranging from 10 to 150 seconds. Once again, this parameter will beexpected to vary with the other operating parameters.

Any suitable apparatus may be employed in connection with the invention.In accordance with highly preferred embodiments of the invention, a jetcooking apparatus is employed. A jet cooking apparatus resembles a jetpump that is employed to move liquids and slurries. In the jet cookingprocess, high pressure saturated steam, at a pressure that ranges fromabout 60 to 200 psig, is injected through a nozzle into the center of aventuri mix combining tube. The slurry is then pulled into the annulargap formed by the steam nozzle and the venturi opening. The slurry isheated as it accelerates to sonic velocity within the mixing tube. Whilepassing through the mixing tube, the cells are subjected to extremelyturbulent conditions which cause partial hydrolysis of the cell walls.

It is contemplated in preferred embodiments of the invention thatmultiple passes through a jet cooking apparatus, preferably between 2 to5 passes, and more preferably 2 to 3 passes, will be employed. If it isdesired to completely liquefy the cells, i.e., to disassociate the cellsto an extent such that the cell walls are substantially completelydissociated with no intact ghosts remaining, a higher number of passes,such as 3 to 7, may be employed. The precise number of passes requiredto achieve complete dissociation and the number of passes required toachieve a mixture of cytoplasm and ghosts will depend upon the specificapparatus employed and on the other operating conditions.

Generally, it is believed that the more aggressive conditions that areemployed, such as higher alkalinity and temperature, the fewer thenumber of passes will be needed to liquefy greater than 90% of thecells. The pH of the slurry will decline after each pass through the jetcooking apparatus, at least because of the introduction of additionalwater via the steam injector, and possibly because of hydroxyl uptake.It is contemplated that additional alkaline agents may be added aftereach pass, but preferably no such agents are added.

The jet cooking may be practiced as a batch process or as a continuousprocess. In either event, the intermediate product formed upon the firstjet cooking pass is preferably held for a retention time ranging from 30seconds to 1 hour. Most preferably, the intermediate product is held ata temperature of 140° to 160° C. and a pressure of 50 to 80 psig, thenflashed to atmospheric pressure before the second or subsequent jetcooking pass. After the final jet cooking step, preferably there is noretention period, although such may optionally be employed. If theproduct is jet cooked over more than two passes, the intermediateproducts prepared after the first pass but before the final pass may beheld for a retention period, or the retention period may be omitted.

In accordance with some embodiments of the invention, a mild, one- ortwo-pass slightly alkaline pretreatment can be employed to slightlydissociate the cells. After such pretreatment, the alkaline liquid canthen be removed, and a slurry of cells formed by adding water. The cellslurry then may be adjusted to the acidic or alkaline conditionsheretofore discussed, and the slurry then may be jet cooked. It iscontemplated that the mild alkaline pretreatment will removecontaminating biomolecules, small metabolites, and related fermentationbroth products that may contribute off-flavors or colors or mayotherwise negatively affect the hydrolyzed cells.

The mixture of cytoplasm and ghosts thus formed is itself deemed to be acommercially valuable product. It is believed that this mixturetypically will have a solids content that ranges from about 17 to 20%,with about 35% of the solids content comprising insoluble materials andthe rest comprising soluble materials. The product mixture thus formedmay be treated in any manner desired. For instance, the soluble portionof the material may be at least substantially separated from theinsoluble portion, such as by centrifugation. The solids material willcomprise largely cell wall ghosts, and the cell wall ghosts may be soldcommercially. The liquid fraction may be further treated, for instance,by spray drying the liquid fraction with a suitable carrier. In someembodiments of the invention, the mixture exiting the jet cooker mayitself be spray dried, with or without a carrier. Any suitable spraydrying carrier may be employed in connection with the invention, such asmaltodextrins, reduced maltodextrins, starches, starch hydrolyzates, andso forth.

The invention contemplates a method for feeding an animal, the methodcomprising feeding the animal a product mixture prepared in accordancewith the foregoing teachings. The animal also may be fed a fraction ofthe mixture heretofore described, for instance, the solids fraction orthe liquid fraction that remains after centrifuging the product mixture.Generally, the animal will be fed an animal feed, which includes themixture heretofore described (or a suitable fraction thereof) incombination with one or more animal nutritive sources. The mixture orfraction prepared in accordance with the present invention may be addedin any amount relative to the other components of the animal feed.Preferably, the mixture or fraction is added in an amount that rangesfrom 0.01 to 25% by weight, although a greater or lesser range is alsocontemplated. The invention is deemed to find particular applicabilityin feeds for swine, ruminants, poultry, and household pets such as catsand dogs, although it is contemplated that the invention may findutility in connection with feeds for other animals. In some embodimentsof the invention, the mixture prepared in accordance with the foregoingteachings, or a fraction of such mixture, may be used in connection withhuman food products. It is believed that the cytoplasm will providenutritive benefit to swine and ruminants, and, surprisingly, it wasfound in one experiment that swine prefer food products prepared inaccordance with the foregoing teachings to similar food productsprepared with a commercially available yeast derivative.

The present invention is deemed to allow the hydrolysis of cell wallswithout the need for mechanical, autolytic, or hydrolytic procedures.Nonetheless, in some embodiments of the invention, autolysis orhydrolysis procedures may be employed in conjunction with the proceduresheretofore described. In such cases, it is contemplated that thedissociation afforded by the invention may decrease incubation time,and/or may improve enzymatic hydrolysis. Although it is not intended tolimit the invention to a particular theory of operation, it is believedthat such other procedures may so operate by exposing additionalproteins, lipids, or carbohydrates on the cell surface. For similarreasons, the dissociation afforded by the invention may be used inconjunction with acid or alkaline hydrolysis procedures by weakening thecell wall prior to such processing.

The invention contemplates the selection of conditions of temperature,pH, and shear to achieve the results desired. By selection ofappropriate conditions, the manner of cell dissolution may be controlledwith precision. For instance, if desired, dissociation of the cell walland release of cytoplasmic components without extensive denaturation ofthe constituent biomolecules may be achieved. Alternatively, if morerigorous conditions are employed, the cell walls may be dissociated toan extent whereby only the robust soluble or insoluble molecules, suchas alkali-insoluble betaglucans, chitin and the like, remain afterprocessing. In some embodiments, the invention may be employed todissociate cell walls and to harvest oligosaccharides that are obtainedtherefrom, with or without rupture of the cell walls.

The following Examples are provided to illustrate the invention butshould not be construed as limiting the scope of the invention.

EXAMPLE 1

This Example illustrates the jet cooking of dried yeast cells in askid-mounted jet cooking pilot scale apparatus.

About 1.8 kg of commercial spray-dried dead Brewer's yeast was added toabout 10 L of cold water with agitation in a mixing tank. After about 5minutes an additional quantity of water was added to bring the finalvolume to about 12 L. The slurry was allowed to mix for another 3 to 5minutes at which time about 500 ml of about 20% concentrated sodiumhydroxide was slowly added to the yeast slurry. The agitation wasadjusted to high speed mixing during and immediately following thealkaline addition. The moisture was allowed to mix at the high speed foranother 5 to 10 minutes whereupon the pH was checked. The measurementshowed that the pH had increased to about 9.2. Another small addition of20% sodium hydroxide was used to increase the pH to about 9.7. Theslurry was allowed to mix for another 3 minutes or so at a high rate ofspeed at which time the agitation was reduced.

The mixing tank, which was an integral component of the skid mounted jetcooker assembly, was connected to a jet cooker by a valve and piping. Atthe appropriate time the valve was opened and the slurry pumped to thecooker. The cooker was calibrated at 320° F. After a residence time inthe jet cooker of about 3 minutes, the slurry exited the cooker and wascollected (Pass 1). After most of the final material had entered thecooker piping, and the mixing tank emptied, the intermediate productthus formed was transferred to the mixing tank and pumped through thecooker again (Pass 2). The sample was then collected as it exited theassembly and set aside to cool.

The mixture thus formed demonstrated clear microscopic confirmation ofcell dissociation as evidenced by the presence of cells wall ghosts.After the first pass through the jet cooker, about 10 to 20% of suchcell wall ghosts were observed. After the second pass, approximately 60to 70% of the cells typically appeared as ghosts.

Some attributes of the processed material are shown in the followingtable. In this table, reported viscosity was measured using a Brookfieldviscometer at room temperature (spindle nos. 1, 2, 3, and 5 were usedfor the respective samples).

Sample PH Viscosity Yeast Slurry 4.3  38 cP Adjusted Slurry 10.0 177 cPJet Cooked Intermediate 9.1 633 cP Product Final Jet Cooked Mixture 8.02230 cP This data suggests that the rupture of the dead yeast cells isaccompanied by a concomitant change in viscosity (increase) and pH(decrease). These changes are interpreted as signaling the pasting anddeterioration of the cell wall, and associated release of wallcomponents such as glucans and oligosaccharides. The release of glucansand oligosaccharides was believed to be responsible for the increase inviscosity.

EXAMPLE 2

A post-distillation fermentation broth from an ethanol production plantwas centrifuged and the solids recovered as a slurry. This slurry wascomposed of about 20% solids which included primarily (80 to 90%) deadBrewer's yeast cells. The slurry was spray-dried in a pilot plant dryerand stored at room temperature. This material was later retrieved fromstorage and processed employing the general parameters and multiple passjet cook procedures outlined in Example 1 and illustrated in FIG. 1using the skid mounted laboratory/pilot plant jet cooking apparatus.

The observations and results of this experiment were consistent withother experiments performed in this manner. Specifically, some (10-20%)dead yeast cell wall dissociation, an increase in viscosity, and adecrease in pH were observed in the first jet cooker pass. Immediatelyfollowing the first pass of the entire sample volume, this material wastransferred back to the jet cooker feed tank and cycled through again.After the second pass, the viscosity increased dramatically, the pHdeclined further and a significant number of the yeast cells appeared asdistended or swollen cell walls. Cell debris which were not particularlyevident in the unprocessed sample or the first pass material were alsoclearly evident.

EXAMPLE 3

This example illustrates the purification of fungal chitin using themethod of the invention and a substantially uniform microbial fungalsource.

A fungal biomass such as Aspergillus niger or Aspergillus oryzae isconcentrated (or dewatered) using a known procedure such as evaporation,centrifugation and the like to about 12 to 17% solids. The pH of theslurry is adjusted to about pH 11 to 12 with about 5 to 10% sodiumhydroxide. The slurry is then jet cooked at 320° F., 50 to 60 psi. Thefirst pass is collected and the pH readjusted to 11 to 12, as needed.This material is then subjected to as many jet cook cycles at thestrongly alkaline pH as required to effect hydrolysis of as much of theprotein, lipids, glucan and other biomolecules as possible. The treatedmaterial is next filtered using vacuum filtration or a related procedureto remove denatured biomolecules and undesirable materials. The filtered(or alkali insoluble) material is washed with water, the pH is adjustedand available for use as a substrate for the production of glucosamineand the like.

EXAMPLE 4

Several thousand pounds of dead brewer's dried yeast obtained from acommercial ethanol distilling operation were treated in accordance withthe present invention to prepare mixtures of cell wall ghosts andcytoplasm. To prepare the mixtures, the following procedures wereemployed.

A 17% yeast slurry was prepared by adding dried dead yeast to water withagitation. After about 10 to 20 minutes of aggressive agitation, the pHof the mixture was adjusted to a pH in the range of 9.5 to 10.0 with 50%NaOH. Two jet cookers were allowed to attain temperatures of about 300°F. The jet cookers were arranged in an in-line configuration.

The yeast slurry was fed into the primary jet cooker at a rate of about1.5 gal/min. The output for this cooker was held for 12 to 15 min. at atemperature of about 150° C. and a pressure of 50 to 80 psig, flashed toambient pressure, then fed directly into the second jet cooker. The jetcooking operations were conducted to maintain a constant flow from theprimary to the secondary jet cooker. The output from the second jetcooker was then collected in a holding vessel. After cooling to atemperature of about 60° to 70° C, the pH of the collected material waslowered from about pH 7 to about pH 4.0 with hydrochloric acid.

A preservative (sodium benzoate) was then added in an amount of 0.8% byweight to minimize microbial growth during transport and storage to aspray drying facility. Approximately 40 to 50 hours after jet cookingwas completed, the product mixture of cell wall ghosts and cytoplasm wasviscous but still fluid product. This product was spray dried in a boxdryer. No additional carrier was employed. The spray dried material wasevaluated and found to have a moisture content of about 5%.

The spray dried mixture was collected and packaged in 50 lb. paper bags.Several hundred such bags were prepared and were stored at roomtemperature.

Samples of the jet cooked material (before spray drying) were collectedthroughout the duration of the run and viewed microscopically. Boththese cooked and those prepared in accordance with the laboratoryprocedure of Example 1 exhibited substantially identical morphology. Itwas found that 80 to 90% of the cells had been ruptured to yield cellwall ghosts. These ghosts were observed to exhibit evidence of cell walldisruption, including distension, loss of rigidity, non uniformity ofsize and shape, and so forth.

EXAMPLE 5

The spray dried mixture prepared in accordance with Example 4 wasanalyzed to yield the following approximate composition. Whole uncookedyeast was also analyzed. It is seen that a portion of the nutritivematerial in the yeast remained substantially unaffected by the jetcooking process, and that the jet-cooked mixture could provide nutritivebenefit.

Dried Yeast Jet cooked (Whole) mixture Protein 45.8 44.57 Fiber, Crude3.6 2.9 Ash 4.5 9.22 Fiber, Dietary, 20.4 19.7 Total Try 0.48 0.48 Cys0.46 0.33 Met 0.69 0.48 Asp 4.56 5.97 Thr 2.11 2.2 Ser 2.25 2.25 Glu6.83 5.77 Pro 2.32 2.48 Gly 1.95 1.87 Ala 3.45 2.98 Val 2.45 2.48 Iso2.09 1.99 Leu 4.03 3.84 Tyr 1.54 1.58 Phe 2.15 2.02 Lys 2.61 2.13 His0.96 0.96 Arg 1.97 1.44 Vit. A 9,350 5,800 Vit B1 7.62 2.78 Vit. B2 11.86.8 Vit. B6 1.56 1.15 Vit. B12 <.002 0.0039 Vit. E <3.0 <3.0 Ca 0.0270.08 Mg 0.16 0.21 P 0.84 1.06 K 0.68 0.83 Na 0.12 2.35 Cu <.0002 0.00051Fe 0.0034 0.006 Mn 10 9.6 Zn 56 46

EXAMPLE 6

Two commercial swine feed formulations, designated herein asformulations A and B, were obtained. The spray dried yeast mixtureprepared in accordance with Example 4 was added to each of thecommercial animal feed formulations to form modified formulations A1 andB1. For comparison, a commercial yeast derivative was also added to eachof the two commercial swine feeds to form modified formulations A2 andB2. The commercial yeast derivative product was composed of cell wallghosts believed to have been prepared by autolysis, in combination withfermentation solubles. The commercial yeast derivative was added to thetwo commercial animal feeds in an amount of 4 lbs. per ton, whereas thespray dried product prepared in accordance with Example 4 was added inan amount of 6 lbs. per ton to achieve a comparable loading of cell wallghosts.

The modified feed formulations A1 and B1 (representing feeds prepared inaccordance with the invention) and A2, and B2 (representing thecomparative product) were fed to 100 pigs. The pigs were housed in tenseparate pens, with ten pigs per pen. Each pen was provided with twofeeders. The modified feed formulations A1 and A2 each were added to oneof the feeders, and the amount of feed consumed by the pigs was measuredafter three days and after six days. The feeders were switched one timeper day in an effort to eliminate any bias that may have been associatedwith the position of the feeder in the pen. In a separate experiment,the modified feed formulations B1 and B2 were added to the feeders, andthe amount of feed consumed by the pigs was measured. The followingresults were observed.

B1 A1 Days Lb/Pig % Lb/Pig % P-Value 0-3 .23 33  .46 67 <.01 0-6 .35 181.60 82 <.01 B2 A2 Days Lb/Pig % Lb/Pig % P-Value 0-3 .12 26  .34 74<.01 0-6 .23 16 1.22 84 <.01

Surprisingly, for both commercial swine feeds, the pigs exhibited astrong preference for the modified feed formulated with the product ofExample 4 relative to the commercial cell wall product. This preferencewas manifest after three days and became more pronounced after six days.These results demonstrate that the pigs exhibited a strong preferencefor the feed that contained the material of Example 4. The improvedpalatability of this feed was seen to enhance feed uptake.

It is thus seen that the invention provides a method for dissociation ofcells.

All references cited herein are hereby incorporated by reference. Allmethods described herein can be performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language provided herein,is intended merely to better illuminate the invention and does not posea limitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-descriedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

1. A method comprising: providing a plurality of microbial cells; andsubjecting said cells to heat, pH, and shear under conditions sufficientto rupture the walls of at least some of said plurality of cells toallow cytoplasm to be released therefrom thereby forming a mixture ofcell wall ghosts and cytoplasm, said cells being cooked in a jet cookingapparatus.
 2. A method according to claim 1, said pH being an alkalinepH.
 3. A method according to claim 2, said pH ranging from 8.5 to 10.5.4. A method according to claim 3, said pH ranging from 9 to
 10. 5. Amethod according to claim 1, said cells being subjected to a temperatureranging from 140 to 160° C.
 6. A method according to claim 1, the methodcomprising; jet-cooking said mixture of cells in a jet-cooking apparatusto form an intermediate mixture; and jet-cooking said intermediatemixture to form said mixture of cell wall ghosts and cytoplasm.
 7. Amethod according to claim 1, said cells comprising yeast cells.
 8. Amethod according to claim 7, said cells comprising S. cereviasiae cells.9. A method according to claim 1, further comprising at leastsubstantially separating said cytoplasm from said cell wall ghosts. 10.A method according to claim 9, said separation being performed bycentrifuging said mixture of cell wall ghosts and cytoplasm.
 11. Amethod according to claim 1, further comprising spray drying saidmixture of cytoplasm and cell wall ghosts.
 12. A method comprising:providing a plurality of microbial cells; empirically determining, forsaid plurality of cells, conditions of heat, shear, and pH sufficient torupture the walls of at least some of the plurality of cells to allowcytoplasm to be released therefrom to thereby yield a mixture of cellwall ghosts and cytoplasm; subjecting said cells to said heat and saidshear and said pH under conditions sufficient to rupture the walls of atleast some of said plurality of cells to allow cytoplasm to be releasedtherefrom and to form a mixture of cell wall ghosts and cytoplasm; saidcells being cooked in a jet cooking apparatus.
 13. A method for thedissociation of microbial cells, comprising: providing microbial cells;dissociating said cells under conditions of pH, temperature, and shearto thereby yield a mixture of dissociated cell wall components, saidconditions having been selected to be suitable for the at least partialdissociation of the walls of said cells, whereby at least one solubledissociated molecular cell wall component is substantially separablefrom the dissociated cell walls, said cells being cooked in a jetcooking apparatus.
 14. A method according to claim 1, said cells beingsubjected to steam.
 15. A method for rupturing microbial cells to yielda mixture comprising cell wall ghosts and cytoplasm, comprising:providing a plurality of microbial cells; and subjecting said cells toheat, pH, and shear under conditions sufficient to rupture the walls ofat least some of said plurality of cells to allow cytoplasm to bereleased therefrom thereby forming said mixture of ghosts and cytoplasm,said cells being subjected to a temperature ranging from 140° to 160°C., the method comprising: jet-cooking and mixture of cells in ajet-cooking apparatus to form an intermediate mixture; and jet-cookingsaid intermediate mixture to form said mixture of cell wall ghosts andcytoplasm.
 16. A method according to claim 15, said cells beingsubjected to steam.